Abstract

Introduction

Both protein-truncating variants and some missense substitutions in CHEK2 confer increased risk of breast cancer. However, no large-scale study has used full
open reading frame mutation screening to assess the contribution of rare missense
substitutions in CHEK2 to breast cancer risk. This absence has been due in part to a lack of validated statistical
methods for summarizing risk attributable to large numbers of individually rare missense
substitutions.

Methods

Previously, we adapted an in silico assessment of missense substitutions used for analysis of unclassified missense substitutions
in BRCA1 and BRCA2 to the problem of assessing candidate genes using rare missense substitution data
observed in case-control mutation-screening studies. The method involves stratifying
rare missense substitutions observed in cases and/or controls into a series of grades
ordered a priori from least to most likely to be evolutionarily deleterious, followed by a logistic
regression test for trends to compare the frequency distributions of the graded missense
substitutions in cases versus controls. Here we used this approach to analyze CHEK2 mutation-screening data from a population-based series of 1,303 female breast cancer
patients and 1,109 unaffected female controls.

Results

We found evidence of risk associated with rare, evolutionarily unlikely CHEK2 missense substitutions. Additional findings were that (1) the risk estimate for the
most severe grade of CHEK2 missense substitutions (denoted C65) is approximately equivalent to that of CHEK2 protein-truncating variants; (2) the population attributable fraction and the familial
relative risk explained by the pool of rare missense substitutions were similar to
those explained by the pool of protein-truncating variants; and (3) post hoc power calculations implied that scaling up case-control mutation screening to examine
entire biochemical pathways would require roughly 2,000 cases and controls to achieve
acceptable statistical power.

Conclusions

This study shows that CHEK2 harbors many rare sequence variants that confer increased risk of breast cancer and
that a substantial proportion of these are missense substitutions. The study validates
our analytic approach to rare missense substitutions and provides a method to combine
data from protein-truncating variants and rare missense substitutions into a one degree
of freedom per gene test.